AIM: To integrally understand the effects of human vascular endothelial cells(VECs) on the proliferation of vascular smooth muscle cells(VSMCs).METHODS: Various kinds of human VECs of different origins were co-cultured with human aortic smooth muscle cells, a representative of human VSMCs. To exclude the irrelevant effects due to growth competition between VECs and VSMCs, the proliferation of VECs had previously been arrested via a low-dose gamma rayirradiation. To discriminately analyze the proliferation of VSMCs from that of VECs, the former cells were labeled with red fluorescent dye while the latter cells were labeled with green fluorescent dye before performing coculture experiments. After 4 d, total cells were harvested and subjected to flow cytometric analyses. Decrements in red fluorescence intensities due to proliferationmediated dilutions were measured and mathematically processed using a specific software to quantitatively evaluate the proliferation of VSMCs. The findings obtained from the flow cytometry-based analyses were further validated by microscopic observations. RESULTS: Commercially available primary cultured human VECs exclusively promoted VSMC proliferation regardless of their tissue origins and we termed these pro-proliferative VECs as “typeⅠ”. By contrast, VECs freshly generated from human bone marrow-derived endothelial progenitors cells or human pluripotent stem cells including embryonic stem cells and induced pluripotent stem cells suppressed VSMC proliferation and we termed these anti-proliferative VECs as “typeⅡ”. Repetitive subcultures as well as oxidative stress induced “type Ⅱ VECs to typeⅠ” conversion along with an induction of Regulator of G-protein signaling 5(RGS5)Compatibly, anti-oxidant treatments suppressed both the subculture-dependent “typeⅡ to typeⅠ” conversion and an induction of RGS5 gene. Immunostaining studies of clinical specimens indicated that RGS5 protein expressions in endothelial layers were low in norma arteries but they were up-regulated in pathologica arteries including hypertension, atherosclerosis and autoimmune vasculitis in a dose-dependent manner Overexpression and knockdown of RGS5 caused that“typeⅡ to typeⅠ” and “typeⅠ to type Ⅱ” phenotype conversions of VECs, respectively. CONCLUSION: Human VECs are categorized into two types: pro-proliferative RGS5~(high) VECs(typeⅠ) and antiproliferative RGS5 ~(low) VECs(typeⅡ). AIM: To integrally understand the effects of human vascular endothelial cells (VECs) on the proliferation of vascular smooth muscle cells (VSMCs). METHODS: Various kinds of human VECs of different origins were co-cultured with human aortic smooth muscle cells, a representative of human VSMCs. To exclude the irrelevant effects due to growth competition between VECs and VSMCs, the proliferation of VECs had previously been arrested via a low-dose gamma ray radiation. labeled with red fluorescent dye while performing latter coculture experiments. After 4 d, total cells were harvested and subjected to flow cytometric analyzes. Decrements in red fluorescence intensities due to proliferationmediated dilutions were measured and mathematically processed using a specific software to quantitatively evaluate the proliferation of VSMCs. The findings ob tained from the flow cytometry-based analyzes were further validated by microscopic observations. RESULTS: Commercially available primary cultured human VECs exclusively promoted VSMC proliferation regardless of their tissue origins and we termed these pro-proliferative VECs as “type I”. By contrast, VECs freshly generated from human bone marrow-derived endothelial progenitors cells or human pluripotent stem cells including embryonic stem cells and induced pluripotent stem cells suppressed VSMC proliferation and wei these anti-proliferative VECs as “type II ”. Repetitive subcultures as well as oxidative stress induced “type II VECs to type I ” conversion along with an induction of Regulator of G-protein signaling 5 (RGS5) Compatibly, anti-oxidant preparations suppressed both the subculture-dependent “type II to type I ” conversion and an induction of RGS5 gene. Immunostaining studies of clinical specimens indicated that RGS5 protein expressions in endothelial layers were low in norma arteries but they were up-regulated in pathologica arteries including hypertension, atherosclerosis and autoimmune vasculitis in a dose-dependent manner Overexpression and knockdown of RGS5 caused that “type II to type I ” and “type I to type II ” phenotype conversions of VECs , respectively. CONCLUSION: Human VECs are categorized into two types: pro-proliferative RGS5 ~ (high) VECs (typeⅠ) and antiproliferative RGS5 ~ (low) VECs (typeⅡ).